Mitochondrial dysfunction is one of the most pervasive and damaging processes that exist in various neurodegenerative diseases and neuropathological conditions. In cultures that were exposed to UV as a stress, the INK pathway has been shown to play critical roles in regulating mitochondrial function. Of the INK isoforms, JNK3 is unique, being enriched in the nervous system and playing critical roles in excitotoxicity- mediated apoptosis. Whether JNK3 is activated under pathological conditions and whether/how JNK3 activation is relayed to mitochondria in vivo, however, have remained unknown. Our preliminary data indicate that JNK3 is selectively activated after spinal cord injury, and regulates injury-mediated cytochrome C (cytC) release and oligodendrocyte apoptosis. The goal of this proposal is therefore to identify critical in vivo targets of JNK3 that execute cytC release, and understand the mechanisms by which JNK3 regulates these molecules to initiate cytC release. We have identified that Mcl-1, a pro- survival Bcl-2 member, is a substrate for JNK3 in vivo. The outcome of this phosphorylation is to facilitate ubiquitin-mediated degradation of Mcl-1, which is normally inhibited by Pinl binding. Pinl, a propyl- isomerase, binds MAP kinase substrates after they are phosphorylated, thereby regulating either the degradation or the activities of the substrates. When Mcl-1 is mutated to render it independent of JNK3 phosphorylation, Pinl fails to dissociate from Mcl-1 under stress and Mcl-1 degradation is delayed. These results together suggest that JNK3-dependent phosphorylation of Mel-1 is necessary for displacing Pin 1, allowing Mcl-1 to be ubiquitinated and degraded so that cytC can be released. Recently, Mule, a BH3-only E3 ubiquitin ligase, was identified being responsible for Mcl-1 ubiquitination and degradation. Our data suggest that Mule binds Mcl-1 in vivo and Pinl may regulate its binding characteristics. We therefore hypothesize that JNK3 and Pinl oppositely regulate ubiquitination of Mcl-1 by Mule, in turn regulating degradation of Mcl-1 and subsequent cytC release after spinal cord injury. Under the hypothesis, we propose the following three specific aims. Aim I is to determine whether Mcl-1 is necessary for cytC release after spinal cord injury by analyzing the conditional null of Mcl-1;Aim II is to determine whether Pinl is necessary for Mcl-1 stabilization and whether JNK3 is involved in Pinl action after spinal cord injury by subjecting Pinl"'" and Pinr^JNKS"'" mice to injury;and Aim III is to determine the molecular mechanisms by which JNK3 regulates Mcl-1 ubiquitination by Mule. We believe these studies will lead to a significant advancement in our understanding of JNK3 action under pathological conditions, and likely to provide novel therapeutic targets for degenerative conditions.